The study was conducted at Egerton University, located in Nakuru County, Kenya. Microbial analyses were performed at the Soil Microbiology Laboratory, while protein analyses were conducted in the Food Analysis Laboratory. Vitamin and antinutrient analyses were carried out at the Animal Science Laboratory, all within Egerton University.

A completely randomized design (CRD) with a factorial arrangement was employed for the study. The main factors investigated included blanching temperature regimes, fermentation time and glucose concentration. Blanching was conducted at temperatures of 80˚C for 10 minutes and 90˚C for 5 minutes. Fermentation durations studied were 0, 7, 14, and 21 days, while glucose concentrations used were 0%, 1%, 2%, and 3%. The experiment was conducted in triplicates. Samples were analyzed for moisture content, protein content, beta-carotene, ascorbic acid, calcium and iron. The six best-performing samples were selected based on their nutritional performance during fermentation for sensory evaluation.

Lactoplantibacillus plantarum, the fermenting microorganism used in this study, was isolated from Mursik, a traditionally fermented milk from the Kalenjin community, Kenya [18] - [20] . Mursik samples were randomly collected from three households and three commercial vendors in Njoro town, Nakuru County, totalling six samples. Approximately 200 ml per sample was obtained using sterile, autoclaved containers. Farmers provided milk in their original fermentation containers, which were mixed thoroughly before sampling. Three replicates per sample were taken aseptically into labelled containers, sealed, and transported at 4˚C to Egerton University within 3 - 4 hours for lactic acid bacteria isolation.

Isolation of LAB for fermentation and sequencing

Isolation of lactic acid bacteria

Lactic acid bacteria were enumerated by pour-plating on de Man, Rogosa, and Sharpe (MRS) agar (Oxoid Ltd.), incubated anaerobically at 35˚C for 2 days using Anaerocult A packs (Merck, Darmstadt, Germany). Distinct colonies from the highest dilution plates were selected based on morphology (size, shape, gloss, colour) using a sterile loop. Colonies were purified by triple streaking on MRS agar and stored in 0.25 mol/L sucrose solution at −18˚C for further identification.

Phenotypic and Physiological Characterization of Lactic Acid Bacteria

Isolates were first confirmed as lactic acid bacteria by Gram staining and catalase testing. Colonies were described morphologically (shape, size, elevation, surface, edges). Gram-positive bacteria stained blue/purple and were either rod-shaped or cocci, while Gram-negative bacteria-stained pink/red under a light microscope. Catalase test involved adding 3% hydrogen peroxide to colonies; gas production indicated a positive result. Only catalase-negative, Gram-positive isolates proceeded to physiological tests. These isolates were cultured in MRS broth to assess growth at 15˚C and 45˚C by observing turbidity after 72 hours. Gas production from glucose metabolism was also evaluated. Salt tolerance was tested by incubating isolates in MRS broth with 4% and 6.5% NaCl at 37˚C for 4 days; increased turbidity indicated positive growth.

Molecular Characterization of Lactic Acid Bacteria

Genomic DNA was extracted from 12-hour-old MRS broth cultures following the method of Cardinal et al. (1997) [21] , with cell lysis achieved using lysozyme, SDS, proteinase K, and CTAB/NaCl, followed by purification with chloroform/isoamyl alcohol. DNA was precipitated with isopropanol, washed with 70% ethanol, air-dried, and resuspended in TE buffer containing RNase. DNA quality and concentration were assessed using a Nanodrop spectrophotometer, and 0.8% of the agarose gel electrophoresis was stained with ethidium bromide.

Purified DNA samples were stored at –20˚C and sent to Inqaba Biotech (South Africa) for 16S rRNA gene sequencing using group-specific primers Lac1 and Lac2-GC, and universal primers 907R(C), 1492R, ITS1, and ITS4. Sequences were analyzed using the BLAST algorithm and aligned in MEGA 6.0 software. Phylogenetic trees were constructed using the neighbour-joining method with 100 bootstrap replicates to assess branch reliability.

Black nightshade and spider plant vegetables were collected from Njoro, Nakuru County. Young and tender leaves were selected and transported to Egerton University for analysis using a cool box at 4˚C. Upon arrival, the leaves were sorted, and the decayed and overmature leaves were discarded. After sorting, the leaves were washed with tap water to remove dirt. Using a sharp sterile knife, the leaves were then shredded into 3 mm pieces. Both raw and processed ILVs were analyzed for moisture content, vitamins (vitamin C, beta-carotene content) and minerals (iron and zinc).

The tender shoots of ILVs were blanched at 80˚C/10mins and 90˚C/5mins [8] . This was performed by bringing a large pan half full of water to a temperature of 80˚C and 90˚C. The shredded vegetables (500 g) were put into a wire basket and gently lowered into blanching water. Once the blanching time was over, the vegetables were removed from the blanching water and plunged into cold, sterile water at room temperature to halt the blanching process.

After blanching, the shredded vegetables were divided into 500g batches for fermentation, with glucose added at concentrations of 0%, 1%, 2%, and 3%. Fermentation was carried out in 2.5% brine, as it is commonly recommended for vegetable fermentation due to its ability to inhibit spoilage microorganisms, support lactic acid bacteria growth, and maintain the texture, flavour, and the final product quality. Subsequently, the vegetables were inoculated with LAB isolates at a concentration of approximately 1 × 10⁸ CFU/mL to achieve this inoculum concentration, a 0.5 McFarland standard was used as a reference. This standard was prepared by mixing 0.05 mL of 1.175% barium chloride with 9.95 mL of 1% sulfuric acid. Well-isolated colonies of LAB previously stored under 2% sucrose were carefully picked and suspended in sterile saline to form a turbid bacterial suspension. The turbidity of this suspension was then visually compared to the 0.5 McFarland standard in a well-lit environment against a white background. The suspension was diluted with sterile saline or concentrated by adding more bacterial cells to match the standard. Additionally, the optical density (OD) at 600 nm was measured using a spectrophotometer to confirm that the suspension fell within the expected OD₆₀₀ range of 0.08 to 0.1, corresponding to the desired bacterial concentration [12] . Fermentation was conducted for up to 21 days at 25˚C, with samples analyzed for moisture content, protein, vitamins (vitamin C, beta carotene), and minerals (calcium and iron) on days (0, 21).

Moisture content was evaluated by oven drying using the Association of Official Analytical Chemists [22] method 970.23. Samples were dried in an oven at 105˚C for 3 hours and then cooled in a desiccator for 10 minutes. Moisture content was calculated as weight loss represented as a percentage of original sample weight.

Using procedure 991.20 of the Kjeldahl technique [22] , the crude protein content was ascertained. Samples (2 grams) were weighed and put into the micro-Kjeldahl digestion tubes. Each tube was then filled with 10 mL of concentrated nitrogen-free sulfuric acid and one selenium tablet, which served as a catalyst. The materials were subsequently digested for three hours at 445 degrees Celsius in a digester. The Kjeldahl distillation apparatus was used to distil the byproducts of digestion. The distillate was collected in 15 millilitres of 0.1M HCl, to which a mixture of methylene blue and methyl red was added as an indicator. The excess HCl was titrated against 0.5 N NaOH. The crude protein content was calculated using Equation (1):

Crude protein (g/100g) = (V₁ − V₂) × M × 1.4 × 6.25/W (1)

where V₂ is the volume of HCl used for the test portion, V₁ is the volume of HCl used for the blank test, M is the molarity of the acid, W is the weight of the test portion, and 6.25 is the conversion factor. The analysis was performed in triplicate, and the results were reported as means ± standard deviations.

Ascorbic acid was determined by titration with 2,6-dichlorophenolindophenol dye following the AOAC method 990.23 [22] . Samples were homogenized in metaphoric acid solution and the extract filtered, then diluted appropriately to a concentration of 100 mg ascorbic acid/100 mL.

A standard solution of pure ascorbic acid was prepared by dissolving 50 mg in 100 mL of water. Sample filtrate was titrated against the standard solution to a pink end point within 10 seconds and ascorbic acid was calculated as per Equation (2):

Concentration of ascorbic acid (mg/g) = C × V × (DF/WT) (2)

where C = Milligrams of ascorbic acid per millilitre of dye (mg/mL).

V = Volume of dye used in titrating the sample (mL).

DF = Dilution factor.

WT = Weight of the sample (grams).

Using spectrophotometry (UV-VIS Spectrophotometer, model 2377, India), the vitamin A content was ascertained in accordance with AOAC International Method 2000.10 [22] . After precisely weighing the sample (2 grams), it was put in a mortar and 15 millilitres of acetone were added. To extract the chlorophyll, the mixture was mashed using a mortar and pestle, and then acid-washed sand was added. After that, 15 mL volumes of acetone were used to rewash the residue until the extract lost its color. Acetone was added to the extract to get it up to the 100 mL level. From this, 25 mL of the extract was evaporated in a rotary vacuum evaporator, and roughly one millilitre of petroleum ether was used to wash the residue. After that, the mixture was put into a silica gel-prepared chromatographic column (TLC Silica Gel 60), and to eliminate any remaining water from the sample, a 1 cm layer of anhydrous sodium sulphate was placed on top of the column. Next, the evaporated samples were quantitatively spotted into a column, dissolved in 2 mL of petroleum ether, and eluted with the petroleum ether. Petroleum ether was used to raise the initial yellow elute to the required level after it had been collected in a 25 mL flask. A CE of 440 UV/Vis double-beam scanning spectrophotometer calibrated with standard solutions of pure beta-carotene in petroleum ether was used to measure the beta-carotene at 450 nm.

A solution of 0.001 g of pure beta-carotene in 1000 mL of petroleum ether was used to prepare the standards. This created a 1000 ppm (1000 µg/mL) solution. The standards were 0.5 µg/mL, 1.0 µg/mL, 1.5 µg/mL, 2.0 µg/mL, 2.5 µg/mL, and 3.0 µg/mL, whose absorbances were read at 440 nm.

The concentration of beta-carotene (CX) was calculated using Equation (3):

$CX\left(\frac{\text{mg}}{100\text{g}}\right)=\frac{AX\times CS\left(\text{mg}/\text{mL}\right)\times \text{Total}\text{volume}\text{of}\text{extract}\left(\text{mL}\right)}{AS\times \text{Sample}\text{weight}\left(\text{g}\right)}$ (3)

where CX represents the concentration of β-carotene, AX represents the peak area of β-carotene, CS represents the concentration of the standard, and AS represents the peak area of the standard.

Using the Atomic Absorption Spectrophotometer (AAS) and the AOAC International technique 970.30 [22] , the two minerals, calcium and iron, were measured. A hollow cathode lamp, a recorder, and an air-acetylene flame were included in the spectrophotometer’s setup. Calcium was measured at a wavelength of 248 nm, and iron was measured at 234 nm. In a digester, 10 mL of concentrated HCl and 20 mL of concentrated HNO₃ were used to digest about 0.5 grams of the material. The samples were heated to 250˚C with caution until the strong reaction stopped. After that, the temperature was raised to 450˚C for 45 minutes. Next, filtering was carried out in 60 mL filter bottles using Whatman filter paper no.4 (22 µm pore size) to obtain the distillate. Distilled water was added until the filtrate reached the desired level. Following a comparison of the amounts of each component with the standards, the calcium and iron concentrations in (mg/100g) were reported.

Panelists who use the product were selected for the test. Prior to the test, the study was reviewed and approved by the Egerton University Research Ethics Committee. An informed consent was obtained from each subject before the sensory evaluation test.

The black nightshade and spider plant vegetables, under different treatments, were conventionally boiled 90˚C/10mins before the sensory test. Spontaneously fermented spider plant and black nightshade vegetables were used as the controls. The six samples were coded and then presented to 30 panelists to evaluate them for appearance, aroma, taste, texture, and overall acceptability. The 5-point hedonic scale was used, with the expressions ranging from “dislike extremely” (1) to “like extremely” (5).

The data obtained from the experiments above were subjected to statistical analysis using statistical package SAS 9.4. The results were expressed as mean ± standard deviation. Means were analyzed for significant differences by analysis of variance (ANOVA) at (p < 0.05)

The nutrient content of fresh black nightshade (BN) and spider plant (SN) per 100 grams is shown in Table 1 . Black nightshade had a statistically higher (p < 0.05) protein content (5.90%), Vitamin A content (11.57 mg/100g), moisture content (87.45%), and Fe (11.33 mg/100g) compared to spider plant (5.61%, 10.97 mg/100g, 83.29%, and 7.12 mg/100g, respectively). Spider plant on its part had statistically higher vitamin C (0.62 g/100g) and calcium (385.69 mg/100g) compared to black night shade (0.52 g/100g and 218.11 mg/100g, respectively). The nutritional values are in the similar range reported in the other studies [23] [24] , with variations attributed to climatic and soil conditions.

Note: BN = Black nightshade; SN = Spider plant; Ptn = Protein; VitC = Vitamin C; VitA = Vitamin A; MC = Moisture content; Ca = Calcium; Fe = Iron. Means followed by the same letter are not significantly different at p < 0.05.

The effect of different blanching regimens on the nutrient content of fermented black nightshade (BN) and spider plant (SN) is shown in Table 2 . In both vegetables, blanching at 80˚C for 10 minutes resulted in significantly higher protein retention (4.81% for BN and 4.03% for SN) compared to blanching at 90˚C for 5 minutes (4.23% for BN and 2.98% for SN) at (p < 0.05). Blanching also led to loss of vitamin A but similar to protein, blanching at 80˚C for 10 minutes led to significantly high vitamin A retention in both vegetables (10.69 mg/100g for BN and 10.18 mg/100g for SN) compared to 90˚C for 5 minutes (9.99 mg/100g for BN and 9.28 mg/100g for SN). Significant differences in moisture content between the two treatments for either vegetable were observed ( Table 2 ). Blanching significantly affected vitamin C content within the two treatments for either vegetable at p > 0.05. Blanching at higher temperatures, 90˚C/5mins led to significant loss of vitamin C (0.48 g/100g for BN and 0.52 g/100g for SN), while blanching at 80˚C /10mins showed a higher vitamin C retention of (0.50g/100g for BN and 0.58 g/100g for SN). Blanching significantly increased the moisture content of both vegetables compared to the fresh vegetables. The results showed a significant decrease in calcium levels (p < 0.05) following blanching, with spider plant exhibiting more pronounced reductions compared to black nightshade. Interestingly, when comparing different blanching conditions within each vegetable type, spider plant blanched at 80˚C for 10 minutes retained significantly high calcium content (241.26 mg/100g) compared to the 90˚C for 5 minutes treatment (229.52 mg/100g) ( Table 2 ). Similarly, blanching led to a significant reduction (p < 0.05) in iron content, with spider plant also demonstrating higher iron retention following milder blanching conditions (3.07 mg/100g for 80˚C/10mins vs. 1.93 mg/100g for 90˚C/5mins). Black nightshade exhibited a similar trend, with higher iron content observed after milder blanching (4.95 mg/100g for 80˚C/10mins vs. 3.53 mg/100g for 90˚C/5mins).

Note: BN = Black nightshade; SN = Spider plant; Ptn = Protein; VitC = Vitamin C; VitA = Vitamin A; MC = Moisture content; Ca = Calcium; Fe = Iron; mins = minutes. Means followed by the same letter are not significantly different at p < 0.05.

The effect of glucose concentration on the nutritional composition of fermented spider plant and black nightshade vegetables was investigated and the results are presented in Table 3 . Both vegetables displayed a trend of increasing protein content with increasing glucose concentration. Black nightshade showed a significant increase in protein content (from 3.92% at 0% glucose to 5.03% at 3% glucose). Spider plant also exhibited a similar trend (from 3.13% at 0% glucose to 4.02% at 3% glucose). Black nightshade displayed a significant increase in vitamin A content with increasing glucose concentration (from 9.52 mg/100g at 0% glucose to 11.09 mg/100g at 3% glucose). Spider plant also showed a similar trend, where vitamin A increased with increase in glucose concentration (from 8.88 mg/100g at 0% glucose to 10.66 mg/100g at 3% glucose). There were no significant differences in vitamin C content observed between different glucose concentrations (1% - 3%) for spider plant vegetables. This suggests that glucose concentration within the

Note: BN = Black nightshade; SN = Spider plant; Ptn = Protein; VitC = Vitamin C; VitA = Vitamin A; MC = Moisture content; Ca = Calcium; Fe = Iron; mins = minutes. Means followed by the same letter are not significantly different at p < 0.05.

tested range (1% - 3%), which influences microbial growth did not significantly affect vitamin C levels. Both vegetables exhibited a slight decrease in moisture content with increasing glucose concentration, with those vegetables with 3% glucose concentration recording significantly low moisture content of 89.76% for BN and 86.30% for SN. Both vegetables showed a significant decrease in Fe²⁺ and Ca²⁺ content with increasing glucose concentration. The lowest iron content of (3.31 mg/100g for BN and 1.89 mg/100g for SN) and calcium content (188.47 mg/100g for BN and 206.30 mg/100g for SN) was recorded for those vegetables with 3% glucose concentration.

The vegetables exhibited a significant increase in protein, vitamin A and vitamin C content with fermentation, as shown in Table 4 . On the other hand, moisture content, Fe²⁺ and Ca²⁺ contents decreased significantly. Fermented black nightshade vegetables recorded the highest levels of protein (5.14%), vitamin C (0.51 g/100g), and vitamin A (10.91 mg/100g) on day 21, while the lowest levels of calcium (122.13 mg/100g), iron (2.03 mg/100g), and moisture content (89.38%) were recorded on day 21. Similarly, fermented spider plant vegetables exhibited the highest levels of protein (3.80%), vitamin C (0.56 g/100g), and vitamin A (10.58 mg/100g) on day 21, while day 21 held the record for the lowest moisture (86.12%), calcium (144.59 mg/100g), and iron (0.79 mg/100g).

Note: BN = Black nightshade; SN = Spider plant; Ptn = Protein; VitC = Vitamin C; VitA = Vitamin A; MC = Moisture content; Ca = Calcium; Fe = Iron; mins = minutes. Means followed by the same letter are not significantly different at p < 0.05.

The study also developed and tested the acceptability of the fermented spider plant and black nigh shade vegetables. Generally, vegetables subjected to controlled fermentation scored preferably higher values based on appearance, aroma, texture, taste and overall acceptability. Whereas the controls (spontaneously fermented) scored the lowest values based on all the attributes tested, as shown in Table 5 . The samples chosen for sensory evaluation were selected based on their nutritional quality following fermentation. Within each blanching regime, the best sample was selected for sensory evaluation. Fermented spider plant vegetables blanched at 90˚C/5mins recorded significantly high levels of aroma, taste and overall acceptability. Whereas fermented black night shade vegetables blanched at 90˚C/5mins scored higher values for appearance and texture. Fermented black night shade and spider plant vegetables blanched at 90˚C/5mins were the most preferred vegetables by consumers ( Table 5 ).

Note: BN = Black nightshade; SN = Spider plant; N. B = Not blanched; S. F = Spontaneously fermented. Means followed by the same letter are not significantly different at p < 0.0.